Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 mug DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Effect of environmental mycobacteria (Mycobacterium avium) on immunity induced by a DNA vaccine (DNAhsp65) against tuberculosis

The efficacy of BCG vaccine (attenuated Mycobacterium bovis) against pulmonary tuberculosis varies enormously among different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment. Studies have revealed that most protective antigens expressed by the antituberculous vaccine are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates a cross-reactive immune response that interferes with BCG efficacy. In this study we investigated the effect of a prior exposure to heat-killed M. avium on the immune response and the protective efficacy induced by a genetic vaccine pVAXhsp65 (hsp65 gene from M. leprae inserted in pVAX vector) against experimental tuberculosis. To evaluate the effect on the immune response, female BALB/c mice were initially injected with distinct doses (0.08x106, 4x106, and 200x106) of heat-killed M. avium by subcutaneous route. Three weeks later, the animals were immunized with 3 doses of DNAhsp65 by intramuscular route (100µg/15 days apart). Control groups received only M. avium, vaccine (pVAXhsp65), vector (pVAX) or saline solution. Cytokine production and antibody levels were determined by ELISA. To evaluate the effect on the protective efficacy, animals were initially sensitized with 200x106 heat-killed CFU of M. avium by subcutaneous route and then immunized with 3 doses of pVAXhsp65 (100µg/15 days apart) by intramuscular route. Control groups were injected with saline, pVAX (4 doses), pVAXhsp65 (4 doses), M. avium or M. avium plus pVAX (3 doses). Fifteen days after last DNA dose, the animals were infected with 1x104 viable CFU of H37Rv M. tuberculosis by intratracheal route. Thirty days after challenge, the animals were sacrificed and the bacterial burden was determined by counting the number of CFU in the lungs. Lung histological sections were also analyzed. Splenic cells from primed animals produced more IL-5 but less IFN-gamma than non-primed ones. Also, prior contact with M. avium determined higher production of IgG1 and IgG2a anti-hsp65 antibodies in comparison to control groups. However, this higher immune response did not decrease the bacterial burden in the lungs. In addition, prior sensitization with M. avium decreased the parenchyma preservation observed in the group immunized only with pVaxhsp65. These results indicate that environmental mycobacteria can interfere with immunity and protective efficacy induced by DNAhsp65.(AU)